Tecnológico Nacional de México/Instituto Tecnológico de Durango, Blvd. Felipe Pescador 1830 Ote. Col. Nueva Vizcaya, 34080, Durango, Dgo., Mexico.
Facultad de Ciencias Químicas, Universidad Juárez del Estado de Durango, Av. Veterinaria S/N, Col. Valle del Sur, 34120, Durango, Dgo., Mexico.
Protein Expr Purif. 2024 Aug;220:106490. doi: 10.1016/j.pep.2024.106490. Epub 2024 May 1.
The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.
木质纤维素生物质中可发酵糖的生产是通过一组称为纤维素酶的酶的协同作用实现的。纤维素是由β-1,4 键化学连接的葡萄糖长链。β-1,4-内切葡聚糖酶是参与降解的第一种纤维素酶，它打破了无定形区域的键。从纸浆和造纸厂废水分离的枯草芽孢杆菌菌株中获得了一种具有高活性的β-1,4-内切葡聚糖酶。测序和生物信息学分析表明，通过 PCR 扩增的基因由 1407 个核苷酸组成，编码约 55 kDa 的β-1,4-内切葡聚糖酶。成功将编码成熟内切葡聚糖酶（eglS）的开放阅读框（ORF）插入修饰后的克隆质粒（pITD03）和用于在酵母中表达的 pYD1 质粒中。羧甲基纤维素（CMC）平板测定、SDS-PAGE 和酶谱分析证实，转化的大肠杆菌 BL21-SI 菌株产生并分泌了一种 39 kDa 的β-1,4-内切葡聚糖酶，与没有纤维素结合模块（CBM）的催化结构域一致。结果表明，截短的β-1,4-内切葡聚糖酶具有更高的活性和稳定性。
